Structural proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are potential drug targets due to their role in the virus life cycle. The envelope (E) protein is one of the structural proteins; plays a critical role in virulency. However, the emergence of mutations oftenly leads to drug resistance and may also play a vital role in virus stabilization and evolution. In this study, we aimed to identify mutations in E proteins that affect the protein stability. About 0.3 million complete whole genome sequences were analyzed to screen mutations in E protein. All these mutations were subjected to stability prediction using the DynaMut server. The most common mutations that were detected at the C-terminal domain, Ser68Phe, Pro71Ser, and Leu73Phe, were examined through molecular dynamics (MD) simulations for a 100ns period. The sequence analysis shows the existence of 259 mutations in E protein. Interestingly, 16 of them were detected in the DFLV amino acid (aa) motif (aa72-aa75) that binds the host PALS1 protein. The results of root mean square deviation, fluctuations, radius of gyration, and free energy landscape show that Ser68Phe, Pro71Ser, and Leu73Phe are exhibiting a more stabilizing effect. However, a more comprehensive experimental study may be required to see the effect on virus pathogenicity. Potential antiviral drugs, and vaccines may be developed used after screening the genomic variations for better management of SARS-CoV-2 infections.

Therapeutic effects of anticancer medicines can be improved by targeting the specific receptors on cancer cells. Folate receptor (FR) targeting with antibody (Ab) is an effective tool to deliver anticancer drugs to the cancer cell. In this research project, a novel formulation of targeting drug delivery was designed, and its anticancer effects were analyzed. Folic acid-conjugated magnetic nanoparticles (MNPs) were used for the purification of folate receptors through a novel magnetic affinity purification method. Antibodies against the folate receptors and methotrexate (MTX) were developed and characterized with enzyme-linked immunosorbent assay and Western blot. Targeting nanomedicines (MNP-MTX-FR Ab) were synthesized by engineering the MNP with methotrexate and anti-folate receptor antibody (anti-FR Ab). The cytotoxicity of nanomedicines on HeLa cells was analyzed by calculating the % age cell viability. A fluorescent study was performed with HeLa cells and tumor tissue sections to analyze the binding efficacy and intracellular tracking of synthesized nanomedicines. MNP-MTX-FR Ab demonstrated good cytotoxicity along all the nanocomposites, which confirms that the antibody-coated medicine possesses the potential affinity to destroy cancer cells in the targeted drug delivery process. Immunohistochemical approaches and fluorescent study further confirmed their uptake by FRs on the tumor cells’ surface in antibody-mediated endocytosis. The current approach is a useful addition to targeted drug delivery for better management of cancer therapy along with immunotherapy in the future.

Genomic characterization of the dengue virus (DENV) is useful for understanding its molecular evolution, transmission, pathogenicity and infectivity. The DENV genomic RNA encodes three structural proteins, capsid (C) envelope (E) and membrane (M) proteins mediating viral entry and assembly during host infection. The current study aims to explore the DENV serotypes and mutations in the E and M proteins. Twenty-three samples of DENV-positive patients were processed and selected for whole genome sequencing (WGS) from the Punjab Province of Pakistan. Among the 23 WGS, 19 samples showed numerous mutations (BioProject ID PRJNA943555). DENV1 and DENV2 are the most prevalent serotypes. A total of 179 mutations were detected in the E protein, in which K203E, T88A, I114L, and I293T are novel. The I270L, T272A, S273L, and T277A were found in the "kl" β-hairpin (aa 270-279). The M protein harbors 74 mutations, of which 24 were novel. Three prominent complementary regions in the prM and E protein complex formations include R6, E46, D47, D63, and D65 on 'pr' peptide, and E84, K64, and H244, K247 on E, remain conserved except R6C. To our knowledge, it is the first comprehensive study of mutations in structural proteins. Genomic epidemiology is critical for analyzing emerging mutations and designing new policies therapeutic efforts for future outbreaks.

Background: Theranostic nanomedicines contain a nanovehicle that has fluorescent properties and can be used for diagnostic, therapeutic and prognostic purposes. The transferrin receptor expression is 1000-fold higher in rapidly growing cancer cells as compared to the normal cells and, therefore, can be used in targeted drug delivery systems. The objective of the present study was to design a novel targeted gold nanoparticle (GNPs)-based theranostic formulation for gastrointestinal (GI) tract-related cancers. The synthesized GNPs were conjugated to transferrin and doxorubicin both separately and collectively to check their cytotoxic properties. The in vitro cytotoxicity of nanocomposites was observed against colon cancer cell line HCT-116. The doxorubicin conjugated nanocomposites showed almost the same cytotoxicity, but more effect at later hours (h). The IC₅₀ and IC₁₀₀ were 50 µg/ml and 250 µg/ml, respectively, equivalent to the doxorubicin weight for GNP theranostic nanomedicine. Results: The maximum effect was observed after 12 h and nanomedicines were still active after 72 h of treatment. Our in vivo data proved that nanomedicine crossed all the barriers and was successfully delivered to the tumour cells. Theranostic nanomedicine’s (TNM) effect on body weight and survival rate on mice was many folds better than mice in pure doxorubicin group. It also showed almost 80% survival rate on day 40. The in vivo and in vitro results show the effects of prolonged drug release and the nanomedicine was not toxic to vital organs of the animal. Conclusion: This is one of its kind studies in which a novel targeted nanomedicines approach was formulated for therapeutic as well as prognostic purposes against GI tract cancer. Graphic

Genomic epidemiology of SARS-CoV-2 is imperative to explore the transmission, evolution, and also pathogenicity of viruses. The emergence of SARS-CoV-2 variants of concern posed a severe threat to the global public health efforts. To assess the potential consequence of these emerging variants on public health, continuous molecular epidemiology is of vital importance. The current study has been designed to investigate the major SARS-CoV-2 variants and emerging mutations in virus structural and non-structural proteins (NSP) during the fourth wave in September 2021 from the Punjab province of Pakistan. Twenty SARS-CoV-2 positive samples have been collected from major cities were subjected to next-generation sequencing. Among the 20 whole genomes (GenBank Accession SRR16294858-SRR16294877), 2 samples failed to be completely sequenced. These genome sequences harbored 207 non-synonymous mutations, among which 19 were unique to GISAID. The genome sequences were detected: Delta 21I, 21J variants (B.1.617.2). Mutation’s spike_F157del, spike_P681R, spike_T478K, spike_T19R, spike_L452R, spike_D614G, spike_G142D, spike_E156G, and spike_R158del have been detected in all samples where K1086Q, E554K, and C1250W were unique in spike protein. These genomic sequences also harbored 129 non-synonymous mutations in NSP. The most common were NSP3_P1469S (N = 17), NSP3_A488S (N = 17), NSP3_P1228L (N = 17), NSP4_V167L (N = 17), NSP4_T492I (N = 17), NSP6_T77A (N = 17), NSP14_A394V (N = 17), NSP12_G671S (N = 18), and NSP13_P77L (N = 18). The mutation, F313Y in NSP12, detected in the current study, was found in a single isolate from Belgium. Numerous other unique mutations have been detected in the virus papain-like protease (NSP3), main protease (NSP5), and RNA-dependent RNA polymerase (NSP12). The most common non-synonymous mutations in the spike protein were subjected to stability analysis, exhibiting a stabilizing effect on structures. The presence of Delta variants may affect therapeutic efforts and vaccine efficacy. Continuous genomic epidemiology of SARS-CoV-2 in Pakistan may be useful for better management of SARS-CoV-2 infections.

Transferrin is an iron binding glycoprotein actively involved in the growth and maintenance of cell cycle. The transferrin receptors expression is increased on growing cancer/tumor cells for absorption of iron through transferrin and participation in biological activity. In this study, a novel method for the purification of transferrin by using magnetic nanoparticles (MNP) is developed and compared with reported method. Magnetic nanoparticles were synthesized by co-precipitation method under hydrothermal conditions in the presence of ammonium hydroxide. MNP were characterized by FTIR, VSM, DLS, TEM, and SEM. Purified transferrin was characterized by SDS-PAGE, MALDI-TOF, ELISA, Western blot, and its activity was further confirmed by iron binding assay and receptor binding assays. Purified transferrin was also conjugated with cysteine capped gold nanoparticles (GNP) and characterized by UV-Vis spectra, TEM, DLS, and fluorescent spectrophotometry. Transferrin conjugated cysteine capped GNP used as a targeted fluorescent probe on gastric cancer, tumor tissue and MDA-MB 231 cancer cells to confirm transferrin receptor binding activity and application as diagnostic probe. The purified transferrin showed stability and activity up to 36 months. The results indicated that the synthesized superamagnetic MNP are good for the purification of transferrin. A good yield of transferrin was purified by this method, good quality and showed active biological activity. GNP conjugated transferrin has a potential to be used in cancer diagnosis as targeted diagnosis probe in vivo and in vitro. Experiments are underway for utilizing transferrin as carrier for targeting drug delivery.